Semipermeable species boundaries permit gene flow but prevent a hybrid swarm among sympatric catostomids of a desert ecosystem.

Interspecific hybridization can reshape patterns of genetic variation and alter evolutionary trajectories, but the implications of naturally occurring hybridization for endangered species remain uncertain. Understanding how species boundaries are maintained or eroded in the presence of hybridization is therefore essential for managing endangered species in altered ecosystems. In the San Juan River, three native catostomids (bluehead sucker Catostomus discobolus, flannelmouth sucker C. latipinnis, and the endangered razorback sucker Xyrauchen texanus) form a hybridizing assemblage in an ecosystem transformed by anthropogenic disturbance. Despite annual reproduction, razorback sucker recruitment is exceptionally rare, whereas adult flannelmouth x razorback sucker hybrids are regularly encountered. We applied double digest restriction site-associated DNA sequencing and mitochondrial DNA barcoding to characterize hybridization dynamics and evaluate whether introgression threatens razorback sucker persistence. Analysis of 30,774 loci from 1128 individuals revealed that 98.8% of hybrids involved crosses between flannelmouth and razorback sucker. F1 (68.4%) and third-generation (28.4%) hybrids were most prevalent, while second-generation hybrids were rarely detected (3.2%). All second and third-generation hybrids resulted from backcrossing. Razorback sucker mitochondrial haplotypes occurred in 92.7% of hybrids, suggesting asymmetric mating or potential mitonuclear incompatibilities. Genomic cline analyses identified loci with non-neutral introgression patterns, including a large chromosomal region consistent with overdominance and others reflecting potential Dobzhanzky-Muller incompatibilities or genetic drift. These patterns, combined with a scarcity of later-generation intermediate hybrids, suggest that disruption of co-adapted gene complexes limits hybridization beyond the F1 generation. Although hybridization occurs regularly, introgression appears constrained, and formation of a hybrid swarm remains unlikely under current conditions.